This application claims priority from copending PCT application PCT/US2004/030986 filed Sep. 9, 2004 and U.S. 60/505,264 filed Sep. 22, 2003. Methods and compositions of multiple splice variants of IG20 are useful to regulate cell death and replication.
Eukaryotes have evolved the process of alternative mRNA splicing for generating multiple protein isoforms from the same gene. It is a highly regulated process that ensures removal of nucleotides at specific locations without disrupting the open reading frame. Alternative mRNA splicing could remove either a whole exon or part of an exon resulting in different transcripts capable of encoding related, but distinct, proteins. Since the completion of the human genome sequencing, it has become apparent that the biological complexity seen in humans, relative to other lower species, is most likely due to a higher degree of alternative splicing in human genes. IG20 is one such gene that undergoes alternative mRNA splicing resulting in the production of multiple proteins.
cDNAs, including that encoding IG20 are differentially expressed in human insulinomas. cDNAs encoding MADD/DENN (MAP Kinase-Activating Death Domain-containing protein/Differentially Expressed in Normal and Neoplastic cells), KIAA0368 and DENN-SV (a short variant of DENN-SV) have near identical sequences, yet different actions.
Over-expression of MADD can enhance MAPK and both ERK and JNK activity, and lead to the phosphorylation of cPLA2 upon TNFα treatment. Additionally, MADD can induce TNFα gene expression and promote TNFα-induced proliferation of Kaposi's sarcoma (KS) cells, which can be inhibited by blocking MADD transcription. The GDP-GTP exchange protein (GEP), a rat homolog of IG20, mediates conversion of GDP-bound inactive form of the Rab-3 subfamily of small G proteins into the GTP-bound active form. Characterization of Rab3 GEP knockout mice showed that the protein is required for vesicle trafficking at the neuromuscular junction and might play a role in the formation of synaptic vesicles Rab3 GEP−/− mice fully develop but die shortly after birth. A related gene with minimal sequence homology to MADD is the AEX gene of the C. elegans. When the above human cDNAs were identified it was not apparent whether they represented products of related but distinct genes or different splice variants arising from a single gene.
Expression studies and sequence comparisons between cDNAs and with human genome data base showed that the above mentioned human cDNAs are splice variants encoded by the IG20 gene. The IG20 gene consists of 36 exons that range in size from 47 to 986 nucleotides, including the 5′ and 3′ un-translated regions. The IG20 splice variants result from alternative splicing of only exons 13L, 16, 21, 26, and 34. Human genome sequencing has revealed that the biological complexity seen in humans, relative to other species, is due to a higher degree of alternative splicing in human genes that could result in multiple proteins with different functions from the same gene.
In an attempt to understand the functional relevance of different splice variants of IG20 gene, HeLa cells were reported because they express all four splice variants, namely, IG20, MADD, IG20-SV2 and DENN-SV, detected to date in human cells and tissues. HeLa cell lines permanently transfected with cDNAs encoding IG20, MADD, and DENN-SV were assessed for TNFα-induced cell death. Consistently, relative to controls, HeLa-IG20 cells were most susceptible and HeLa-DENN-SV cells were most resistant to TNFα-induced cell death. Results obtained with HeLa-MADD cells were comparable to those obtained with HeLa cells transfected with an empty vector.
To understand why cells transfected with different splice variants of IG20 gene responded differently to TNFα treatment, potential functional motifs in the spliced regions were sought. An extensive search failed to reveal any apparent functional domains in the spliced regions. This suggested that splicing most likely results in conformational changes that affect their cellular localization or interactions with other proteins. Upon treatment with TNFα, all variants could interact with TNFR1 and enhance ERK activation. Differences in response to TNFα treatment of cells transfected with different IG20 splice variants are most likely not due to differences in these properties.
TNFα induced cell death is mediated through recruitment of FADD to the TNFR1/TRADD complex and activation of initiator caspase 8. Activation of caspase 8 leads to the activation of effector caspase 3 that cleaves a wide range of substrates ultimately leading to cell death. Upon treatment with TNFα/CHX, HeLa-IG20 cells showed maximal caspase activation. As expected, HeLa-Vector and HeLa-MADD cells showed only a moderate activation of caspase 8. Surprisingly, HeLa-DENN-SV cells showed little, if any, caspase 8 activity upon identical treatment. Consistent with these data, higher and lower levels of cleaved-active caspase 8 and caspase 3 were noted in HeLa-IG20 and HeLa-DENN-SV cells, respectively, compared to control cells. Activation of caspases was critical by inhibiting cell death in the presence of CrmA, which preferentially inhibits caspases 1 and 8 and thus can block activation of caspase 3 and prevent TNFα-induced cell death. IG20 appears to be acting primarily upstream of caspase-8 and its interaction with TNFR1 can enhance TNFα-induced caspase 8 activation. This also indicated that enhancing caspase activation is the dominant function of IG20 since it can override its own up-regulation of ERK activation commonly associated with cell survival.
Although the IG20 gene can encode multiple splice variants that are functionally different, how many are naturally expressed in various human tissues was not known prior to the present disclosure. Whether splice variants (e.g. DENN-SV) that are highly expressed in tumors contribute to enhanced cell proliferation and/or resistance to cell death was unknown.
IG20 and IG20-VB2, and the previously reported KIAA0358, MADD, and DENN-SV are splice variants of the IG20 gene, which is localized to chromosome 11p11 and consists of 36 exons. Differences among the above variants are due to alternative splicing of exons 13L, 16, 21, 26 and 34. Cell transfection studies showed that IG20 and DENN-SV conferred susceptibility and resistance respectively, to TNFα-induced apoptosis, whereas, MADD expression had no discernible effect. All three variants interacted with tumor necrosis factor type 1 (TNFR1) and enhanced activation of the extracellular-regulated kinase (ERK), but only IG20 enhanced activation of caspases 8 and 3. Further, IG20-mediated, TNFα-induced apoptosis could be abrogated by the caspase inhibitor, CrmA. These results suggested that enhancement of apoptosis by IG20 is mainly dependent on activation of caspases 8 and 3. In addition, several studies have implicated a role for IG20 splice variants in tumor formation. However, to date, no systematic study has been conducted to determine which variants are naturally expressed in human tumors and whether they might influence tumor cell growth and/or susceptibility to various treatments leading to induced cell death.
The tumor necrosis factor (TNF) super family of ligands and receptors play a critical role in the regulation of organogenesis, homeostasis, inflammation, innate, and adaptive immunity. A subset of TNF family ligands can bind to their cognate death receptors on cells and activate apoptosis. Inappropriate regulation of apoptosis could result in chronic inflammation, autoimmunity, or development of cancer.
TNF Related Apoptosis Inducing Ligand (TRAIL) can selectively kill some cancer cells and render others susceptible to co-treatment with drugs and irradiation, with little or no effect on most normal cells. TRAIL induced apoptosis is of considerable interest and has significant implications for developing novel cancer therapies.
The TNF Related Apoptosis Inducing Ligand (TRAIL) is a unique member of the TNF super family that can kill cancer cells selectively with little or no effect on most normal cells. Recombinant TRAIL, when systemically administered, can result in tumor shrinkage in vivo and in some cases, their complete elimination without resulting in any of the adverse systemic side effects often associated with TNF-α or CD95L. TRAIL, when used alone, can kill some tumor cell lines, however, its efficacy, when used in combination with chemotherapy and γ-irradiation, is high.
TRAIL can bind to 5 distinct receptors—Death Receptor 4 (DR4 or TRAILR-1), Death Receptor 5 (DR5 or TRAILR-2), Decoy Receptor 1 (DcR1, TRAILR-3, LIT or TRID), Decoy Receptor 2 (DcR2, TRAILR-4, TRUNDD) and Osteoprotegerin (OPG). Among these receptors, only DR4 and DR5 contain cytoplasmic Death Domains (DD) and are able to transduce apoptotic signals upon TRAIL binding. Although TRAIL can ligate both the DcR1 and DcR2, since their cytoplasmic tails lack a DD or have a partial DD, they are unable to transduce apoptotic signals. Little is known about OPG except that it is a soluble receptor and its association with TRAIL is relatively weak. Similar to TNFα and CD95L induced signaling, upon TRAIL treatment, FADD and caspase-8 are recruited to the DR4 and DR5 death inducing signaling complexes (DISC). Thus TRAIL; TNF-α and CD95L induced apoptotic signaling pathways share some common features. Earlier, the ability of TRAIL to selectively kill cancer cells was attributed to the dominant negative effects of the decoy receptors (DcRs) expressed on normal, but not on cancer cells, that can compete for TRAIL binding. More recent studies have shown that factors other than DcR expression confer resistance to TRAIL induced cell death.
IG20, MADD and DENN-SV could increase activity of TNF-α induced Mitogen Activated Protein (MAP) Kinase and Extracellular Signal Regulated (ERK) Kinase. Their ability to promote apoptosis however, varied. The IG20 and the DENN-SV splice variants rendered cells more susceptible and resistant to apoptosis respectively, whereas, the MADD splice variant had little or no apparent effect. Additional studies showed that all the three splice variants could interact with the TNFR1 upon TNF-α treatment, but only HeLa cells transfected with IG20 splice variant showed enhanced activation of caspase-8 and -3 that could be blocked by CrmA. IG20 acts as a pro-apoptotic molecule in enhancing TNF-α induced apoptosis.
IG20 can directly interact with TNFR1 and TRADD, but not CD95 and FADD indicating a potential role in TNFR1, but not CD95, mediated signaling. Analyses of DD sequences from different adaptor proteins showed that the DD of IG20 is more homologous to the DDs of DR4 and DR5 than it is to the DD of TNFR1 and TRADD.
The contrasting effects of DENN-SV and IG20 on susceptibility to death inducing stimuli suggests that the eventual outcome of these signaling pathways in tumor cells is determined, at least in part, by a balance in the expression levels of these two proteins. Indeed, HeLa cells that normally express all 4 variants, upon treatment with TNF-α undergo apoptosis, however, approximately only one half of the cells die. When dying cells were separated, on the basis of expression of apoptotic markers, from living cells and tested for expression of various splice variants, it was noted that while cells undergoing apoptosis expressed higher levels of the IG20 the viable cells expressed higher levels of DENN-SV.
Radiation therapy takes advantage of the inherently unstable nature of tumors. The DNA lesions induced by γ-irradiation activate an intrinsic cellular pathway for dealing with DNA damage. Cells initiate a set of physiological responses thought to facilitate DNA repair processes that include cell cycle arrest in G1, S phase, and G2, a slowing of DNA replication, and increased transcription of genes encoding proteins that participate in DNA replication and repair. If the degree of damage suffered by a cell is extensive, then the apoptotic pathway is activated leading to cell death. At the molecular level, several pathways have been studied, including p53 dependent and p53 independent pathways. Other molecules involved in the response to DNA damage include ATM, ATR, DNA-PK, hCds1/Chk2 and p21. Molecular mechanisms that regulate DNA damage, remain unclear.